Profiling microRNAs in lung tissue from pigs infected with Actinobacillus pleuropneumoniae.

BMC Genomics. 2012 Sep 6;13:459.

Podolska A, Anthon C, Bak M, Tommerup N, Skovgaard K, Heegaard PM, Gorodkin J, Cirera S, Fredholm M.

Department of Veterinary Clinical and Animal Sciences, Section of Anatomy, Cell Biology, Genetics and Bioinformatics, University of Copenhagen, Faculty of Health and Medical Sciences, Copenhagen, Denmark.



MicroRNAs (miRNAs) are a class of non-protein-coding genes that play a crucial regulatory role in mammalian development and disease. Whereas a large number of miRNAs have been annotated at the structural level during the latest years, functional annotation is sparse. Actinobacillus pleuropneumoniae (APP) causes serious lung infections in pigs. Severe damage to the lungs, in many cases deadly, is caused by toxins released by the bacterium and to some degree by host mediated tissue damage. However, understanding of the role of microRNAs in the course of this infectious disease in porcine is still very limited.

In this study, the RNA extracted from visually unaffected and necrotic tissue from pigs infected with Actinobacillus pleuropneumoniae was subjected to small RNA deep sequencing. We identified 169 conserved and 11 candidate novel microRNAs in the pig. Of these, 17 were significantly up-regulated in the necrotic sample and 12 were down-regulated. The expression analysis of a number of candidates revealed microRNAs of potential importance in the innate immune response. MiR-155, a known key player in inflammation, was found expressed in both samples. Moreover, miR-664-5p, miR-451 and miR-15a appear as very promising candidates for microRNAs involved in response to pathogen infection.

This is the first study revealing significant differences in composition and expression profiles of miRNAs in lungs infected with a bacterial pathogen. Our results extend annotation of microRNA in pig and provide insight into the role of a number of microRNAs in regulation of bacteria induced immune and inflammatory response in porcine lung.

PMID: 22953717




Mature microRNAs are single stranded RNAs of 18-25nt in length constituting a highly abundant class of non-coding RNAs discovered over a decade ago. MicroRNAs provide a new exciting mechanism of gene regulation at post transcriptional level by down-regulating their target mRNAs. Over the last few years thousands of microRNAs have been discovered in different organisms including plants and animals and the conservation of most of the microRNAs has been confirmed across species.

Biogenesis of microRNAs is a complex and multi-step process starting in the nucleus with transcription of microRNA genes and their processing. This is followed by the export to the cytoplasm, enzymatic processing by Dicer and incorporation into RISC complex (miRNA induced silencing complex) to obtain functional mature microRNA.

In animals, the interaction between the RISC incorporated microRNA and its target mRNA is based on partial complementarity of the two sequences (the microRNA and the mRNA). The main aim of microRNA is to inhibit protein synthesis by means of mRNA cleavage based degradation or by inhibition of translation (Figure 1).

Susanna Cirera Salicio-Picture1 png

Figure 1: microRNA biogenesis. Modified from de Planell-Saguer and Rodicio (2011).


It appears that about 60% of human protein coding genes host microRNA recognition sites mostly in their 3’ UTR. This highly extensive range of targets gives a huge number of possibilities to regulate protein coding genes in various biological scenarios including development, cell proliferation and differentiation, homeostasis, apoptosis and disease. In fact, one of the most exciting aspects of microRNA biology is their contribution to the very complex developmental regulatory networks. Today it is known that many microRNAs are expressed in developmental stage-specific or tissue-specific manner pointing towards their importance in regulatory networks responsible for developmental timing, early embryonic development, organogenesis or maintenance of homeostasis.

Actinobacillus pleuropneumoniae infection in pigs

Pigs are commonly raised by farmers for meat production. These animals are rather prone to infectious diseases like the one caused by Actinobacillus pleuropneumoniae (APP). Actinobacillus pleuropneumoniae is a non-motile, gram-negative bacterial pathogen causing respiratory disease in porcine throughout the world. Indeed, the contagious pleuropneumonia causes severe economic losses to the pig industry worldwide (Figure 2).

Susanna Cirera Salicio Picture2

Figure 2: Sampling areas of the porcine lungs infected with Actinnobacillus pleuropnemoniae.

1 – Necrotic area, within pulmonary lesion.

2 – Demarcation zone, border area between visually unaffected and pulmonary lesions.

3 – Visually unaffected area, which was sampled either from unaffected lung lobe or from site as distant from pulmonary lesion as possible.


Despite the number of studies evaluating factors involved in the infection there is still a considerable lack of knowledge about the full picture of inflammatory response to APP infection. Studies performed so far, concentrate on protein coding genes, thus omit the very important aspect of a possible role of microRNAs in pig inflammatory response to this serious pathogen.

The lethality as a consequence of APP infection in pigs is primarily caused by shock-like condition developed when inflammatory and immunological responses are disproportionate and cytokines are being released uncontrollably. Such a dramatic disorder leading to death could be influenced by the aberrant expression of microRNAs involved in host response to pathogen i.e miR-21, miR-146a, miR-155. This possible explanation is based upon studies in other bacterial infections and demands validation in the pig infected with APP. It is certainly of high interest and relevance to study the microRNA expression in APP infected pigs and combine this knowledge with the already existing information about microRNA expression in infectious diseases as well as the repertoire of possible microRNA targets already discovered in APP infection.

More studies on porcine microRNAs are required for expansion of the repertoire of these small regulatory elements involved in development, growth and pathological conditions. And the more is known about the host response to the pathogen, the better understanding of the pathogenesis and molecular mechanisms of the immunological response to the infection.

The importance of our study

Total RNA was extracted from necrotic and visually unaffected lung tissue of artificially APP infected individuals and subjected to small RNA high throughput sequencing. Reads were aligned to the porcine genome and microRNAs were annotated. The most significant candidates differently expressed between unaffected and necrotic lung samples were validated by quantitative real-time PCR.

We identified 168 conserved and 11 candidate novel microRNAs in the pig. A total of 32 out of 179 miRNAs were up regulated in the necrotic sample. The expression patterns of 14 candidates and their predicted targets revealed a number of very interesting interactions between our microRNA candidates and major factors regulating immunological response and inflammation. In particular, miR-155 a known key player in inflammation was also found differentially expressed in our study. Moreover, miR-664-5p, miR-451 and miR-15a appear as very good candidates for microRNAs of high importance in response to pathogen infection.

 This is to our knowledge the first study revealing significant differences in miRNA expression profiles between bacterial infected and non-infected areas of lungs. The study sheds light on microRNAs that are of potential importance for regulation of immune and inflammatory responses in AP infection in pigs. A number of the microRNAs we have identified have not been described as regulators of immune system previously which points towards their possible organ specific, infection time specific or species specific role. Some of our results might, however, be of general importance to bacterial infections in other tissues and in other species. This study contributes to better understanding of the complex microRNA regulatory role of the immune response and may contribute to drug improvements in the future.



de Planell-Saguer M, Rodicio MC.Analytical aspects of microRNA in diagnostics: a review.

Anal Chim Acta. 2011 Aug 12;699(2):134-52.


This work was supported by grant from High Technology Foundation and PhD grant from Faculty of Life Sciences, KU to AP. The bioinformatics work was supported by the Danish Council for Independent Research (Technology and Production Sciences), The Danish Council for Strategic Research (Programme Commission on Strategic Growth Technologies), as well as the Danish Center for Scientific Computing.

Susanna Cirera & Merete Fredholm

Department of Veterinary Clinical and Animal Sciences, Section of Genetics, Breeding and Bioinformatics, University of Copenhagen, Faculty of Health and Medical Sciences, Copenhagen, Denmark.


Agnieszka Podolska

University of Copenhagen, BRIC, Copenhagen, Denmark.

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